Mucopenetrating formulations

ABSTRACT

Disclosed herein are compositions and kits comprising a mucopenetrating substance, a therapeutic nucleic acid, and a cationic polymer in an amount sufficient to charge neutralize the therapeutic nucleic acid. Also provided herein are methods of using and producing the compositions and kits.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 63/048,617, filed Jul. 6, 2020 which isincorporated by reference herein in its entirety.

BACKGROUND

Delivery of a therapeutic molecule to the gastrointestinal tract isreferred to as enteric delivery, which may include oral delivery,gastric delivery, or rectal delivery. Therapeutic molecules for entericdelivery are formulated with the intention of achieving a high level ofabsorption of the therapeutic molecule from the intestine, through theintestinal wall, and into circulating blood to achieve systemicdelivery. Therapeutic molecules are usually absorbed from the intestineby passive transfer, which includes diffusion of a molecule through thelipid cell membrane of the epithelial cells lining the inside ofintestines. Factors taken into consideration when formulatingtherapeutic molecules for enteric delivery (e.g., oral, gastric, orrectal delivery) include ionization and lipid solubility of themolecule, gastrointestinal motility, splanchnic blood flow, and moleculesize.

SUMMARY

Provided herein are compositions comprising therapeutic nucleic acidsthat are formulated to traverse the mucus layer covering the epithelialcell lining of the gastrointestinal (GI) tract and, in some embodiments,to traverse the epithelial cell lining. The present disclosure is based,at least in part, on experimental data demonstrating that the mucuslayer forms a barrier that prevents charged molecules (such astherapeutic nucleic acids) from traversing the GI tract lining.Surprisingly, the data provided herein shows that where conventionalpermeability enhancers e.g., fatty acids, fail, the combination ofmolecular charge neutralization and mucopenetrating substance(s)succeeds. The formulations provided herein permit efficient andeffective delivery of therapeutic nucleic acids and other closelyrelated charged compounds through the mucus layer and lining of the GItract.

Some aspects of the present disclosure provide compositions comprising amucopenetrating substance, a therapeutic nucleic acid, and a cationicpolymer in an amount sufficient to charge neutralize the therapeuticnucleic acid.

Other aspects of the present disclosure provide cells comprising acomposition that includes a mucopenetrating substance, a therapeuticnucleic acid, and a cationic polymer in an amount sufficient to chargeneutralize the therapeutic nucleic acid.

Further aspects of the present disclosure provide complexes produced bycombining a mucopenetrating substance with a therapeutic nucleic acid,and a cationic polymer in an amount sufficient to charge neutralize thetherapeutic nucleic acid.

Yet other aspects of the present disclosure provide methods comprisingdelivering to a subject a mucopenetrating substance, a therapeuticnucleic acid, and a cationic polymer in an amount sufficient to chargeneutralize the therapeutic nucleic acid.

Still other aspects of the present disclosure provide methods fordecreasing gene expression in a subject, comprising delivering to amucosal surface of a subject a composition described herein, in aneffective amount to decrease gene expression in a cell in a local regionof the mucosal surface.

Some aspects of the present disclosure provide methods forsynergistically decreasing gene expression in a subject, comprisingdelivering to a mucosal surface of a subject a C10 fatty acid and acomposition as described herein, in an effective amount tosynergistically decrease gene expression in a cell in a local region ofthe mucosal surface, optionally wherein the composition furthercomprises the C10 fatty acid.

In some embodiments, a mucopenetrating substance comprises a non-ionicemulsifier. A mucopenetrating substance, in some embodiments, hasmucolytic activity and/or mucotransport activity.

In some embodiments, a therapeutic nucleic acid, and a cationic polymerform a complex through ionic interactions. The complex, in someembodiments, further comprises the mucopenetrating substance.

In some embodiments, a cationic polymer is a linear polymer. In someembodiments, a cationic polymer is a branched polymer.

In some embodiments, a cationic polymer comprises a cationic lipid. Forexample, a cationic polymer may be selected from the group consistingof: polyquaternium, PDMAEMA (poly(2-dimethylaminoethyl methacrylate),MADQUAT (poly(2-(trimethylamino)ethyl methacrylate)), polyallylamines,polyvinylamines, polyethylenimine, polylysines, cationic polyaminoacids,and cationic polysaccharides. In some embodiments, a cationic polymer isselected from the group consisting of: polyallylamines,polyethyleneimines, and polylysines.

In some embodiments, the cationic polymer is a polyethyleneimine, forexample, a branched polyethyleneimine. In some embodiments, thepolyethyleneimine has a molecular weight of about 5-30 kilodaltons (kDa)or about 10-25 kDa (e.g., about 10-20, about 10-15, about 15-25, about15-20, about 10, about 15, about 20, or about 25 kDa).

In some embodiments, the cationic polymer is a polyallylamine. In someembodiments, the polyallylamine has a molecular weight of lower thanabout 50 kDa, or 50 kDa or lower (e.g., about 5-50, about 10-50, about15-50, about 20-50, about 25-50, about 5-40, about 10-40, about 15-40,about 20-40, about 25-40, about 5-30, about 10-30, about 15-30, about20-30, about 5-25, about 10-25, about 15-25, about 20-25, about 25-25,about 5, about 10, about 15, about 20, about 25, about 30, about 35,about 40, about 45, or about 50 kDa). In some embodiments, thepolyallylamine has a molecular weight of lower than about 40 kDa, lowerthan about 30 kDa, or lower than about 20, kDa.

In some embodiments, the cationic polymer is a polylysine. In someembodiments, the polylysine has a molecular weight of about 10-55 kDa orabout 15-50 kDa (e.g., about 20-50, about 25-50, about 30-50, about35-50, about 40-50, about 45-50, about 15-40, about 20-40, about 25-40,about 30-40, about 35-40, about 15-30, about 20-30, about 25-30, about15-25, about 20-25, about 15, about 20, about 25, about 30, about 35,about 40, about 45, or about 50 kDa).

In some embodiments, the concentration of cationic polymer in thecomposition is about 5-35 mg/ml or about 10-30 mg/ml (e.g., about 15-30,about 20-30, about 25-30, about 10-20, about 15-20, about 10-15, about10, about 15, about 20, about 25, or about 20 mg/ml).

In some embodiments, a non-ionic emulsifier is selected from the groupconsisting of: polysorbates, poloxamers, polyoxylglycerides,macrogolglycerol ricinoleate, polyethylene monostearate, sorbitanmonoesters and triesters, substituted polyethylene glycols, andderivative thereof. In some embodiments, the non-ionic emulsifier iscaprylocaproyl polyoxyl-8 glyceride (LABRASOL®), polysorbate 40 (TWEEN®40), polysorbate 80 (TWEEN® 80), macrogolglycerol ricinoleate(KOLLIPHOR® P188), or oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6glyceride)(LABRAFIL®).

In some embodiments, the concentration of non-ionic emulsifier in thecomposition is about 5-45 mg/ml or about 10-40 mg/ml (e.g., about 15-40,about 20-40, about 25-40, about 30-40, about 35-40, about 10-30, about15-30, about 20-30, about 25-30, about 10-20, about 15-20, about 10-15,about 10, about 15, about 20, about 25, about 20, about 25, about 30,about 35, or about 40 mg/ml).

In some embodiments, a mucopenetrating substance is selected from thegroup consisting of: bromohexine, L-cysteine methylester, bromalein,ambroxol, guaifenesin, and N-acetyl L-cysteine, and dornase alfa. Insome embodiments, a mucopenetrating substance is bromalein or decanoicacid.

In some embodiments, a mucopenetrating substance is bromalein and/or thenon-ionic emulsifier is oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6glyceride)(LABRAFIL®).

In some embodiments, a nucleic acid is deoxyribonucleic acid (DNA) orribonucleic acid (RNA). A nucleic acid may have, for example, aphosphorothioate backbone. In some embodiments, the nucleic acid isdouble-stranded or single-stranded. A nucleic acid, in some embodimentscomprises a modification, optionally a chemical modification and/or agenetic modification. In some embodiments, the nucleic acid has a lengthof 10 to 50 nucleotides. In some embodiments, the nucleic acid is not alocked nucleic acid (LNA) or a peptide nucleic acid (PNA).

In some embodiments, a therapeutic nucleic acid is selected from thegroup consisting of antisense oligonucleotides and RNA interferencemolecules. For example, the RNA interference molecules may be selectedfrom the group consisting of short-hairpin RNAs (shRNAs),small-interfering RNAs (siRNAs), and micro RNAs (mRNAs).

In some embodiments, a therapeutic nucleic acid is an antisenseoligonucleotide (ASO).

In some embodiments, a therapeutic nucleic acid targets SMAD7 mRNA. Forexample, a therapeutic nucleic acid may be mongersen (GED-0301).

In some embodiments, a cationic polymer and a therapeutic nucleic acidare present at a ratio of at least 1:1, at least 5:1, or at least 10:1cationic polymer:therapeutic nucleic acid.

In some embodiments, compositions comprise an antisense oligonucleotide(ASO), polyethylenimine (PEI), and optionally a mucopenetratingsubstance, wherein the PEI is present in an amount sufficient to chargeneutralize the therapeutic nucleic acid.

In some embodiments, compositions comprise an antisense oligonucleotide(ASO) (e.g., mongersen (GED-0301)), poly(2-(trimethylamino)ethylmethacrylate) (MADQUAT), and optionally a mucopenetrating substance,wherein the MADQUAT is present in an amount sufficient to chargeneutralize the therapeutic nucleic acid.

In some embodiments, a composition comprising a mucopenetratingsubstance, a therapeutic nucleic acid, and a cationic polymer is in asolution, is lyophilized, or is in the form of a tablet, optionally withan enteric coating.

In some embodiments, a composition is a pharmaceutical compositionfurther comprising a pharmaceutically-acceptable excipient.

In some embodiments, a therapeutic nucleic acid is an engineered nucleicacid, optionally a recombinant nucleic acid or a synthetic nucleic acid.

In some embodiments, delivery of a mucopenetrating substance, atherapeutic nucleic acid, and a cationic polymer is to a mucosal surfaceof the subject (e.g., orally, gastrointestinal tract, rectal tissue, orvaginal tissue).

In some embodiments, gene expression in a subject is reduced by at least20% relative to gene expression in a subject relative to gene expressionin a subject who has not received the composition or has received acomposition comprising the therapeutic nucleic acid without the cationicpolymer and/or the mucopenetrating substance.

In some embodiments, a subject has a gastrointestinal disorder and/orhas a compromised gastrointestinal barrier. For example, agastrointestinal disorder may be an inflammatory bowel disorder. In someembodiments, the inflammatory bowel disorder is irritable bowel syndrome(IBS), ulcerative colitis, or Crohn's disease.

In some embodiments, transport of the therapeutic nucleic acid throughthe mucosal surface is at least 5-fold, at least 10-fold, at least15-fold, or at least 20-fold higher than uptake of a therapeutic nucleicacid without the cationic polymer and/or the mucopenetrating substance.

The present disclosure, in some aspects, also provides multiple wellplates, wherein wells of the plates comprise a receiver chamberunderlying a permeable membrane onto which a mucus layer has beendeposited.

Also provided herein, in some aspects, are methods for assessingmucotransport of a substance, comprising applying the substance to awell, and assessing transport of the substance through the mucus layer.In some embodiments, a substance comprise a mucopenetrating substance.

Other aspects of the present disclosure provide a composition comprisingan antisense oligonucleotide (ASO), non-ionic emulsifier, and a cationicpolymer, wherein the composition comprises the cationic polymer in anamount sufficient to charge neutralize the ASO.

In some embodiments, the cationic polymer is selected frompolyallylamine (PALL), polylysine (PLL), and polyethyleneimine (PEI).For example, the cationic polymer may be PALL. In some embodiments, thePALL has a molecule weight of lower than 50 kilodaltons (kDa). Forexample, the PALL has a molecular weight of about 10-20 kDa, optionallyabout 15 kDa. As another example, the cationic polymer may be PLL. Insome embodiments, the PLL has a molecule weight of about 15-50 kDa. Asyet another example, the cationic polymer may be PEI. In someembodiments, the PEI has a molecule weight of about 10-25 kDa. In someembodiments, the cationic polymer is branched. In some embodiments, theconcentration of the cationic polymer in the composition is about 10-30mg/ml.

In some embodiments, the non-ionic emulsifier is selected from oleoylpolyoxyl-6 glyceride/oleoyl macrogol-6 glyceride (LABRAFIL®), PluronicF127, polysorbate 40 (TWEEN® 40), polysorbate 80 (TWEEN® 80), andKolliphor P188. In some embodiments, the concentration of the non-ionicemulsifier is about 10-40 mg/ml.

In some embodiments, the cationic polymer is PALL, optionally having amolecule weight of below 50 kDa, and the non-ionic emulsifier isselected from oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6 glyceride(LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN® 40), polysorbate 80(TWEEN® 80), and Kolliphor P188.

In some embodiments, the cationic polymer is PLL, optionally having amolecule weight of about 15-50 kDa, and the non-ionic emulsifier isselected from oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6 glyceride(LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN® 40), polysorbate 80(TWEEN® 80), and Kolliphor P188.

In some embodiments, the cationic polymer is PEI, optionally branchedPEI, optionally having a molecule weight of about 10-25 kDa, and thenon-ionic emulsifier is selected from oleoyl polyoxyl-6 glyceride/oleoylmacrogol-6 glyceride (LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN®40), polysorbate 80 (TWEEN® 80), and Kolliphor P188.

Other aspects of the present disclosure provide a composition comprisinga therapeutic nucleic acid, a non-ionic emulsifier, and a cationicpolymer having a molecular weight of 50 kDa or lower, wherein thecomposition comprises the cationic polymer in an amount sufficient tocharge neutralize the ASO. In some embodiments, the cationic polymer hasa molecular weight of about 10-50 kDa, about 15-50 kDa, or about 10-25kDa. In some embodiments, the therapeutic nucleic acid is an antisenseoligonucleotide (ASO).

Yet other aspects of the present disclosure provide a compositioncomprising an ASO, non-ionic emulsifier, and a zwitterionic polymer. Insome embodiments, the zwitterionic polymer is polyvinylpyrrolidine. Insome embodiments, the polyvinylpyrrolidine has a molecular weight ofabout 50-100 kDa.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein. It is to be understood that thedata illustrated in the drawings in no way limit the scope of thedisclosure.

FIG. 1 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers at different concentrationscomplexed to FAM-Mongersen. Results are summarized as a heatmaps thatshows fold change relative to non-formulated Mongersen. White colorindicates fold changes higher than 4-fold.

FIG. 2 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with non-ionic emulsifiers at different concentrations. Resultsare summarized as a heatmaps that shows fold change relative tonon-formulated Mongersen. White color indicates fold changes higher than15-fold.

FIG. 3 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with mucolytic agents at different concentrations. Results aresummarized as a heatmaps that shows fold change relative tonon-formulated Mongersen. White color indicates fold changes higher than20-fold.

FIGS. 4A-4B show Least Squares Means Plots, which show relative changein tissue permeability and apical tissue accumulation of FAM-Mongersenusing different molecular weight branched polyethyleneimine polymers.The results are based on a statistical regression analysis using 6different non-ionic emulsifiers combined withpolyethyleneimine-Mongersen polyplex.

FIG. 5 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with additional excipients: Non-ionic emulsifiers (KolliphorP188, Labrafil, Tween 40, Tween 80), mycolytic (bromalein) orpermeability enhancer/mucodisruptor (decanoic acid). FAM fluorescenceintensity was quantified by fluorescence microscopy analysis of tissuecross-sections. The results show fold change relative to non-formulatedMongersen.

FIG. 6 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with non-ionic emulsifiers including Kolliphor P188, Poloxamer407, Labrafil, Tween 20, Tween 40, Tween 60, Tween 80. FAM fluorescenceintensity was quantified by fluorescence microscopy analysis of tissuecross-sections. The results show fold change relative to non-formulatedMongersen.

FIG. 7 shows tissue uptake in esophagus, stomach, jejunum, colon andrectum of various formulations consisting of cationic polymers complexedto FAM-Mongersen combined with non-ionic emulsifiers (Labrasol and Tween40). FAM fluorescence intensity was quantified by fluorescencemicroscopy analysis of tissue cross-sections. The results show foldchange relative to non-formulated Mongersen in the corresponding tissuesegment.

FIG. 8 shows tissue uptake of various formulations consisting ofcationic polymers complexed to FAM-Mongersen combined with non-ionicemulsifiers (Labrasol and Tween 40) in jejunum. FAM fluorescenceintensity was quantified by fluorescence microscopy analysis of tissuecross-sections of biopsy samples harvested from jejunum segments exposedto formulations in pigs in vivo. The results show fold change relativeto non-formulated Mongersen in the corresponding tissue segment.

FIGS. 9A-9B show effect of charged surfactants on tissue uptake offormulations. FIG. 9A shows tissue uptake of various formulationsconsisting of cationic polymers complexed to FAM-Mongersen combined withnon-ionic emulsifiers (Labrasol and Tween 40) in jejunum. FAMfluorescence intensity was quantified by fluorescence microscopyanalysis of tissue cross-sections of biopsy samples harvested fromjejunum segments exposed to formulations in pigs in vivo. FIG. 9B showsthe data in FIG. 9A in the form of a heatmap as show fold changerelative to non-formulated Mongersen in the corresponding tissuesegment.

FIGS. 10A-10B show IHC analysis of the fluorescence signal of Famlabeled Mongersen in vivo in pigs in the jejunum, where the Mongersen istreated with various Mongersen-polyplex formulations. effect of chargedsurfactants on tissue uptake of formulations.

FIGS. 11A-11B shows uptake of FAM-Mongersen into apical (FIG. 11A) andbasal (FIG. 11B) intestinal tissue.

FIGS. 12A-12B shows uptake of FAM-Mongersen into apical (FIG. 11A) andbasal (FIG. 11B) intestinal tissue for various formulations with andwithout mucolytics.

FIG. 13 shows transport through native porcine mucus obtained from thejejunum of various FAM-Mongersen formulations. Microdiffusion iscalculated by measured FAM fluorescence intensity in receiver chambercompared to the initial donor fluorescence intensity after 1 hour ofincubation.

FIG. 14 shows apical jejunum tissue uptake of various formulationsconsisting of cationic polymers complexed to Cy5-siRNA combined withnon-ionic emulsifiers.

FIG. 15 shows apical jejunum tissue uptake of various formulationsconsisting of cationic polymers complexed to Cy3 conjugated plasmid DNAcombined with non-ionic emulsifiers.

FIG. 16 shows a Least Squares Means Plot of the relative change inapical tissue accumulation of FAM-Mongersen using different molecularweight branched polyethyleneimine polymers. The results are based on astatistical regression analysis using six (6) different non-ionicemulsifiers combined with polyethyleneimine-Mongersen polyplex.

FIG. 17 shows the average apical tissue accumulation of FAM-Mongersenusing different molecular weight polyallylamine polymers combined withfour (4) different non-ionic emulsifiers. Values are expressed as foldchange compared to the non-formulated FAM-Mongersen control.

FIG. 18 shows the average apical tissue accumulation of FAM-Mongersenusing different molecular weight polylysine polymers combined with four(4) different non-ionic emulsifiers. Values are expressed as fold changecompared to the non-formulated FAM-Mongersen control.

FIG. 19 shows the average apical tissue accumulation of FAM-Mongersenusing different concentrations of polyallyllamine 15 kDa and non-ionicemulsifiers Kolliphor P188 and TWEEN® 80. Results are summarized as abar graph that shows fold change relative to Mongersen in PBS buffer.

DETAILED DESCRIPTION

Mucus is a viscoelastic and adhesive gel that has evolved to protect thegastrointestinal (GI) tract, lung airways, vagina, eye, and othermucosal surfaces by rapidly trapping and removing foreign particles andhydrophobic molecules. See Lai S K et al. Adv Drug Deliv Rev. 2009;61(2): 158-171, incorporated herein by reference. Mucus is composedprimarily of crosslinked and entangled mucin fibers secreted by gobletcells and submucosal glands. Mucins are large molecules (e.g., 0.5-40MDa in size) formed by the linking of numerous mucin monomers (e.g.,0.3-0.5 MDa in size), and are coated with proteoglycans. In addition tomucins, mucus gels are loaded with cells, bacteria, lipids, salts,proteins, macromolecules, and cellular debris. The various componentswork together to form a nanoscopically heterogeneous environment forparticle transport. Mucus viscoelasticity is tightly regulated inhealthy subjects by controlling the mucin to water secretion ratio, aswell as by varying lipid, protein, and ion content. See Lai S K et al.2009.

The limited permeability of drug delivery particles and many hydrophobicdrugs through the mucus barrier leads to their rapid clearance from thedelivery site, often preventing effective biomolecular and drugtherapies at non-toxic dosages. A number of diseases could be treatedmore effectively and with fewer side effects if therapeutic substancescould be more efficiently delivered to the underlying mucosal tissues ina controlled manner. See Lai S K et al. 2009.

To avoid the rapid mucus clearance mechanism and/or reach the underlyingepithelia, therapeutic substances must quickly traverse at least theoutermost layers of the mucus barrier (that is cleared most rapidly).Mucus layer thickness depends strongly on anatomical site, and can rangefrom less than 1 micron up to several hundred microns. To penetratemucus, therapeutic substances, such as therapeutic nucleic acids, mustavoid adhesion to mucin fibers and be small enough to avoid significantsteric inhibition by the dense fiber mesh. See Lai S K et al. 2009.Further, the heterogeneity of mucus (e.g., within an individual orrelative to two individuals) introduces variation in themucopenetrability of therapeutic nucleic acids.

Therapeutic nucleic acids are nucleic acids (or closely relatedcompounds) used to treat disease. See Sridharan K and Gogtay N J Br JClin Pharmacol. 2016 September; 82(3): 659-672, incorporated herein byreference. Although there are various types of therapeutic nucleicacids, they share a common mechanism of action that is mediated bysequence-specific recognition of endogenous nucleic acids throughWatson-Crick base pairing. Their development as therapeutic substanceshas specific distinct requirements because they fall somewhere betweensmall molecules and biologics. Therapeutic nucleic acids are chargedsubstances with physicochemical properties different from small moleculedrugs and can be unstable in a biological environment. Further, nucleicacids typically have to be delivered to the correct intracellularcompartment to have a therapeutic benefit. See Sridharan K and Gogtay NJ 2016.

Both antisense oligonucleotides (ASOs) and aptamers are being exploredas therapeutic nucleic acids. ASOs are single, short-stranded sequences(e.g., 8-50 base pairs in length) that bind to a target mRNA by means ofstandard Watson-Crick base pairing. After an ASO binds with the mRNA toform a target complex, either the target complex will be degraded byendogenous cellular RNase H or a functional blockade of mRNA occurs dueto steric hindrance. Aptamers are single-stranded synthetic DNA or RNAmolecules (e.g., 56-120 nucleotides in length) that bind with highaffinity to the nucleotides coding for proteins and thus serve asdecoys. DNA aptamers are short single-stranded oligonucleotide sequenceswith very high affinity for the target nucleic acids through structuralrecognition. See Sridharan K and Gogtay N J 2016.

RNA interference (RNAi) molecules are also being explored as therapeuticnucleic acids. RNAi is a process by which RNA molecules with sequencescomplementary to a gene coding sequence induce degradation of thecorresponding messenger RNAs (mRNAs) thus blocking the translation ofmRNA into protein. Therapy with siRNA thus has great potentialapplication for diseases caused by abnormal expression or mutation suchas cancers, viral infections and genetic disorders as RNA interferencecan be experimentally triggered. siRNAs are ‘short’ double-strandedmolecules (e.g., 21-23 nucleotides long) and generally can be chemicallysynthesized. siRNAs have the advantage over DNA oligonucleotides in thatthey are always delivered as duplexes, which are more stable. Two majorissues with siRNAs relate to their off-target effects and delivery intothe cell. See Sridharan K and Gogtay N J 2016.

Various strategies for delivering charged therapeutic nucleic acids,successfully are being evaluated. Among these strategies include the useof cationic polymers and cationic lipids. Cationic polymers (includingco-polymers) are often used to induce DNA condensation because they formstrongly charged complexes with the anionic phosphate groups located onthe DNA backbone. The resulting complexes can protect nucleic acids fromenzymatic degradation and facilitate cellular entry. Cationic lipidsform cationic liposomes that electrostatically bind to anionic nucleicacids, forming complexes (lipoplexes) that are taken up into cells byendocytosis. See Sasaki Y et al. Colloid and Interface Science inPharmaceutical Research and Development, 2014.

Despite the ongoing research involving various therapeutic deliverystrategies, delivery of charged therapeutic nucleic acids, to the GItract remains a challenge. The present disclosure provides, inter alia,compositions for effective delivery of charged therapeutic nucleic acidsto the GI tract. In some embodiments, the compositions include amucopenetrating substance, a therapeutic nucleic acid, and a cationicpolymer in an amount sufficient to charge neutralize the therapeuticnucleic acid.

Mucopenetrating Substances

Compositions of the present disclosure, in some embodiments, comprise a(at least one) mucopenetrating substance. A mucopenetrating substance isa substance that facilitates the transport (penetration) of atherapeutic molecule through a mucus layer. It should be understood thatthe effects of a mucopenetrating substance on a therapeutic molecule canbe assessed relative to a control condition, such as delivery (e.g.,across a mucus layer and/or into the GI tract) of the particulartherapeutic molecule in the absence of a particular mucopenetratingsubstance. In some embodiments, a mucopenetrating substance facilitatesthe transport of a therapeutic molecule through both the mucus layer andan underlying epithelial layer, such as the epithelial lining of the GItract. A mucopenetrating substance may, for example, be formulated witha therapeutic molecule such that it binds to the therapeutic moleculethrough covalent or non-covalent interactions. In some embodiments, amucopenetrating substance associates with a therapeutic molecule throughelectrostatic interactions. In some embodiments, such as those in whichthe therapeutic molecule is a nucleic acid, the mucopenetratingsubstance intercalates the nucleic acid (e.g., inserts between basepairs of DNA—see, e.g., work by Leonard Lerman discussed in NucleicAcids in Chemistry and Biology, 3^(rd) Ed. Blackburn G M et al., RSCPublishing, 2006).

The mucus layer can prevent a therapeutic molecule from penetratingthrough the mucus layer through several different mechanisms. Forexample, a therapeutic molecule may associate with (e.g., bind tothrough non-covalent interactions) chyme and/or mucin fibers of themucus layer and be targeted for excretion (see, e.g., Lai S K et al.(Adv Drug Deliv Rev. 2009; 61(2): 158-171). Thus, a mucopenetratingsubstance of the present disclosure, in some embodiments, facilitatesthe transport of a therapeutic molecule through the mucus layer byinhibiting the association between the therapeutic molecule and thechyme and/or the mucin fibers of the mucus layer.

Other mechanisms by which mucopenetrating substances can facilitate thetransport of a therapeutic molecule (e.g., a therapeutic nucleic acid)through the mucus layer and/or underlying epithelial lining includetransient opening of tight junctions in the epithelial lining,disruption of lipid bilayer packing in the epithelial lining, and/oraltering the fluidity of the intestinal epithelial lining.

In some embodiments, a mucopenetrating substance improves passivetransport of a therapeutic molecule through a mucus layer and/orunderlying epithelial lining. In some embodiments, a mucopenetratingsubstance improves active transport of a therapeutic molecule through amucus layer and/or underlying epithelial lining.

In some embodiments, a mucopenetrating substance increase the rate atwhich a therapeutic molecule traverses a mucus layer and/or underlyingepithelial layer. For example, the rate at which a therapeutic moleculetraverses a mucus layer and/or underlying epithelial layer whenformulated with a mucopenetrating substance may increase by at least10%, at least 20%, at least 30%, at least 40%, or at least 50%, relativeto the therapeutic molecule not formulated with the mucopenetratingsubstance. In some embodiments, the rate at which a therapeutic moleculetraverses a mucus layer and/or underlying epithelial layer whenformulated with a mucopenetrating substance increases by 10%-50%,20%-50%, 30%-50%, 40%-50%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, or50%-100%. In some embodiments, the rate at which a therapeutic moleculetraverses a mucus layer and/or underlying epithelial layer whenformulated with a mucopenetrating substance increases by at least 100%,or at least 200%.

In some embodiments, a mucopenetrating substance increase the amount ofa therapeutic molecule that traverses a mucus layer and/or underlyingepithelial layer. For example, the amount of a therapeutic molecule thattraverses a mucus layer and/or underlying epithelial layer whenformulated with a mucopenetrating substance may increase by at least10%, at least 20%, at least 30%, at least 40%, or at least 50%, relativeto the therapeutic molecule not formulated with the mucopenetratingsubstance. In some embodiments, the amount of a therapeutic moleculethat traverses a mucus layer and/or underlying epithelial layer whenformulated with a mucopenetrating substance increases by 10%-50%,20%-50%, 30%-50%, 40%-50%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, or50%-100%. In some embodiments, the amount of a therapeutic moleculetraverses a mucus layer and/or underlying epithelial layer whenformulated with a mucopenetrating substance increases by at least 100%,or at least 200%.

In some embodiments, a mucopenetrating substance decreases the clearancerate (excretion) of a therapeutic molecule from the mucus layer. Forexample, the clearance rate of a therapeutic molecule when formulatedwith a mucopenetrating substance may decrease by at least 10%, at least20%, at least 30%, at least 40%, or at least 50%, relative to thetherapeutic molecule not formulated with the mucopenetrating substance.In some embodiments, the clearance rate of a therapeutic molecule whenformulated with a mucopenetrating substance decreases by 10%-50%,20%-50%, 30%-50%, 40%-50%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, or50%-100%. In some embodiments, the clearance rate of a therapeuticmolecule when formulated with a mucopenetrating substance decreases byat least 100%, or at least 200%.

Mucolytic Substances

Mucopenetrating substances, in some embodiments, have mucolyticactivity. This type of mucopenetrating substance is referred to hereinas a mucolytic substance. Mucolytic substances alter the physicalproperties (e.g., viscosity) of a mucus layer in a way that facilitatestransport of a therapeutic agent through the mucus layer. In someembodiments, a mucolytic substance exhibits enzymatic activity. Thereare several classes of mucolytic substances, including classicmucolytics, peptide mucolytics, and nondestructive mucolytics.

Classic mucolytics depolymerize the mucin glycoprotein oligomers byhydrolyzing the disulfide bonds that link the mucin monomers. Classicmucolytics typically contain sulfhydryl groups. A non-limiting exampleof a classic mucolytics is N-acetyl L-cysteine (NAC).

Peptide mucolytics depolymerize the DNA polymer (dornase alfa) or theF-actin network (e.g., gelsolin, thymosin β4). A non-limiting examplesof a peptide mucolytics is dornase alfa (PULMOZYME™).

Nondestructive mucolytics are substances that “loosen” the polyionictangled network of mucin that is formed by charged oligosaccharide sidechains. Examples of nondestructive mucolytics include, but are notlimited to, low-molecular-weight dextran and heparin.

Non limiting examples of mucolytic substances that may be used asprovided herein include bromohexine, L-cysteine methylester, bromalein,ambroxol, guaifenesin, and bromohexine.

Non-Ionic Emulsifiers

The compositions of the present disclosure, in some embodiments,comprise a (at least one) non-ionic emulsifier. In some embodiments, anon-ionic emulsifier is also a mucopenetrating substance. An emulsifieris a substance that can stabilize an emulsion, which is a mixture of twoor more liquids that are otherwise immiscible. Emulsifiers generallykeep molecules from precipitating out of a solution by providinghydrophobic groups onto which hydrophobic areas of the molecules canassociate, thus preventing them from associating with other moleculesand forming larger particles that are likely to leave the solution.Emulsifiers also typically have hydrophilic groups which keep themsoluble in aqueous solutions of moderate to high ionic concentrations.Emulsifiers, natural or synthetic, can be nonionic, anionic, cationic,or amphoteric.

Non-ionic emulsifiers have no overall charge. In some embodiments, anon-ionic emulsifier comprises a hydrophilic portion that includes freehydroxyl and oxyethylene groups and a lipophilic portion havinglong-chain hydrocarbons of fatty acids and fatty alcohols. Non-limitingexamples of natural non-ionic emulsifiers include fatty acid alcohols(e.g., stearyl alcohol and cetyl alcohol), wool fat or wool wax and itsderivatives, wool alcohols and cholesterol, and derivatives of othernatural waxes (e.g., such as spermaceti and cetyl esters wax (syntheticspermaceti). Non-limiting examples of synthetic non-ionic emulsifiersinclude complex esters and ester-ethers, derived from polyols, alkyleneoxides, fatty acids, and fatty alcohols.

Other non-limiting examples of non-ionic emulsifiers includecaprylocaproyl polyoxyl-8 glyceride (LABRASOL®), polysorbate 40 (TWEEN®40), macrogolglycerol ricinoleate (KOLLIPHOR® P188), or oleoylpolyoxyl-6 glyceride/oleoyl macrogol-6 glyceride (LABRAFIL®).

Therapeutic Nucleic Acids

Therapeutic nucleic acids are nucleic acids (or closely relatedcompounds) used to treat disease. See Sridharan K and Gogtay N J Br JClin Pharmacol. 2016 September; 82(3): 659-672, incorporated herein byreference. Treatment herein refers to a reduction in the frequency orseverity of at least one sign or symptom of a disease or disorderexperienced by a subject. The compositions described herein aretypically administered to a subject in an effective amount, that is, anamount capable of producing a desirable result. The desirable resultwill depend upon the active agent being administered. For example, aneffective amount of a composition comprising a therapeutic molecule(e.g., a therapeutic nucleic acid) may be an amount of the compositionthat is capable of causing a desirable expression of a gene or reductionin the expression of a gene in a host organ, tissue, or cell. Atherapeutically acceptable amount may be an amount that is capable oftreating a disease, e.g., a disease of the GI tract. As is well known inthe medical and veterinary arts, dosage for any one subject depends onmany factors, including the subject's size, body surface area, age, theparticular composition to be administered, the active ingredient(s) inthe composition, time and route of administration, general health, andother drugs being administered concurrently.

A therapeutic nucleic acid may be single-stranded or double-stranded. Insome embodiments, a therapeutic nucleic acid may comprise one or moresegments that are single-stranded and one or more segments that aredouble-stranded.

A therapeutic nucleic acid may be a DNA (e.g., a DNA based on antisenseoligonucleotides or DNA aptamers) or an RNA (e.g., microRNAs, shortinterfering RNAs, ribozymes, RNA decoys, and circular RNAs). In someembodiments, a therapeutic nucleic acid is a DNA-RNA hybrid, having bothDNA and RNA segment(s). A therapeutic molecule, in some embodiments,comprises a backbone that is different than that of a DNA or RNA. Forexample, a therapeutic nucleic acid may have a phosphorothioatebackbone.

In some embodiments, a therapeutic nucleic acid is an antisenseoligonucleotide. As discussed above, an antisense oligonucleotide is asingle, short-stranded sequence (e.g., 8-50 base pairs in length) thatbinds to a target mRNA by means of standard Watson-Crick base pairing.See, e.g., Rinaldi C and Wood M. Nature Reviews Neurology 2018; 14:9-21). For example, mongersen (GED-0301) is an antisense oligonucleotideused to block the transcription of RNA encoding SMAD7 protein.

In some embodiments, a therapeutic nucleic acid is a RNA interferencemolecule (e.g., a short-hairpin RNA (shRNA), a small-interfering RNAs(siRNA), or a micro RNA (mRNA)). As discussed above, RNAi is a processby which RNA molecules with sequences complementary to a gene codingsequence induce degradation of the corresponding messenger RNAs (mRNAs)thus blocking the translation of mRNA into protein. See, e.g., Setten Ret al. Nature Reviews Drug Discovery 2019; 18: 421-446.

In some embodiments, a therapeutic nucleic acid has a length of 10-100nucleotides. For example, a therapeutic nucleic acid may have a lengthof 10-75, 10-50, 10-25, 15-100, 15-75, 15-50, 15-25, 20-100, 20-75,20-50, 25-100, 25-75, or 25-50 nucleotides. In some embodiments, atherapeutic nucleic acid has a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Insome embodiments, a therapeutic nucleic acid is longer than 100nucleotides.

A therapeutic nucleic acid may be an engineered nucleic acid, whichincludes synthetic and recombinant nucleic acids. Synthetic nucleicacids are nucleic acids that are made by chemical synthesis. Recombinantnucleic acids are nucleic acids that are made using recombinanttechnologies (e.g., genetic recombination, and by having an organismsuch a bacteria make the desired nucleic acid).

Therapeutic nucleic acids may be modified. Non-limiting examples ofnucleic acid modifications include addition of one or more deoxyinosine,deoxyuridine, amino dU, 2-aminopurine, 5-bromodeoxycytidine,5-bromodeoxyuridine, aminohexyl (aminolink), phosphate, thiol,hexaethylene glycol, thiophosphate, 5-iododeoxyuridine, and5-methyldeoxycytidine. In some embodiments, modifications are made tothe sugar phosphate backbone, e.g., a Phosphorothioate backbone or a2′-O-methyl backbone. In some embodiments, a therapeutic nucleic acid isformulated as a salt (e.g., a sodium salt). A modified therapeuticnucleic acid may or may not be a locked nucleic acid (LNA) or a peptidenucleic acid (PNA). A modification of a therapeutic nucleic acid mayalso be a genetic modification (e.g., an amino acid substitution).

In some embodiments, a therapeutic nucleic acid is conjugated to a smallmolecule or a protein or peptide (e.g., to target a particular cell typeor tissue, or a dye molecule for detection purposes). In someembodiments, a nucleic acid is encapsidated or engulfed by a lipidsubstance.

In some embodiments, a therapeutic nucleic acid modulates (increases ordecreases) expression of a molecular target for an inflammatory boweldisorder (e.g., disease). For example, a therapeutic nucleic acid maytarget any one or more of the molecules described by Katsanos K H andPapadakis K A Gut Liver 2017; 11(4): 455-463, incorporated herein byreference. Non-limiting examples of molecule targets include apoptoticmolecules (e.g., caspase-8), Toll-like receptors (e.g., TLR-4),macrophages (TGFβ, TNF-α, IFN-γ, cytokines [IL-6, IL-9, IL-12, IL-23]),dendritic cells, defensins, regulatory T cells, T effector cells (Th1,Th2, Th17), B cells, dendritic cells, Smad7, JAK inhibitors (e.g.,tofacitinib), adhesion molecules (e.g., MAdCAM-1), anti-integrins (e.g.,anti-α4β7), genes involved in innate mucosal defense and antigenpresentation (NOD2, MDR1, PPAR-γ), redox-sensitive signaling pathwaysand proinflammatory transcription molecules, dendritic cells,adipocytes, fibroblasts, and myofibroblasts.

Cationic Polymer

Data provided herein shows that charge neutralizing an otherwisenegatively-charged therapeutic molecule (e.g., a therapeutic nucleicacid) by formulating it with a cationic polymer i.e., 10-25 kDa and amucopenetrating substance facilitates transport of an effective amountof the therapeutic molecule through both the mucus layer and theepithelial lining of the GI tract. Cationic macromolecules (e.g.,cationic polymers) are macromolecules that have an overall positivecharge. The positive charges of the cationic macromolecules may beattributed to either the macromolecular backbone, side chains, or boththe backbone and the side chains. In some embodiments, a cationicmacromolecule is naturally occurring. In other embodiments, a cationicmacromolecule is synthetic (not naturally occurring). In someembodiments, a cationic macromolecule is a cationic polymer. A polymeris a macromolecule composed of repeated subunits. The role of cationicpolymers in drug delivery systems is discussed by Farshbaf et al. inArtificial Cells, Nanomedicine, and Biotechnology 2018; 46(8): 1872-1891and Samal et al. in Chem Soc Rev. 2012; 41(21): 7147-94, each of whichis incorporated herein by reference). In some embodiments, a cationicpolymer is a cationic lipid.

Charge neutralization refers to a state in which the net electricalcharge of particles, fibers, colloidal material, and/or polyelectrolytesin aqueous solution have been canceled by the adsorption of an equalnumber of opposite charges. This may be characterized as a Zetapotential measurement. For example, as provided herein, an otherwisenegatively charged nucleic acid may be complexed with a cationicmacromolecule is an amount sufficient to charge neutralize the nucleicacid. That is, the overall negative charge of the nucleic acid iscanceled by the positive charges of the cationic macromolecule.

In some embodiments, a cationic macromolecule is linear. In otherembodiments, a cationic macromolecule is branched (e.g., PAMAMdendrimers).

In some embodiments, a cationic polymer is a cationic gelatin. In someembodiments, a cationic polymer is a cationic chitosan (e.g., chitosanlow MW, chitosan high MW, chitosan medium MW). In some embodiments, acationic polymer is a cationic cellulose. In some embodiments, acationic polymer is a cationic dextran. See, e.g., Farshbaf et al. 2018.

Non-limiting examples of cationic polymers includepoly(2-N,N-dimethylaminoethylmethacrylate) PDMAEMA, poly-L-lysine (PLL),poly(ethyleneeimine) (PEI), poly(amidoamine) (PAMAM), chitosan (e.g.,low, medium, or high MW), dL-Lysine monohydrochloride,polydiallyidimethyl ammonium, polyethylenimine (e.g., of 25,000 MW, or800 MW), ply 2-ethyldimethylammoinoethylmethacrylate ethylsulfate-co-1-vinylspyrolidone (having an average MW of 1,000,000, alsoreferred to herein as MADQUAT), poly 2-dimethylaminoethylmethacrylatemethylchloride, poly L-Lysine hydrobromide (e.g., MW 1000-5000,15000-25000, 5-15000, and 30000-70000), PLKC, mPEGK-b-PLKC50, andPLCK-PEG5K-b-PLK50. Any one or more of the preceding cationic polymersmay be combined with an otherwise negatively-charged therapeutic nucleicacid in an amount sufficient to charge neutralize the therapeuticnucleic acid.

Complexes

Provided herein, in some embodiments, are complexes comprising atherapeutic molecule and a cationic macromolecule. A complex, in someembodiments, further comprise a (at least one) mucopenetratingsubstance. In some embodiments, a therapeutic molecule and a cationicmacromolecule (and/or a mucopenetrating substance) form a complexthrough non-covalent interactions, such as ionic interactions.

In some embodiments, a therapeutic molecule (e.g., a therapeutic nucleicacid) as provided herein is complexed with a cationic macromolecule. Insome embodiments, a therapeutic molecule (e.g., a therapeutic nucleicacid) and a cationic macromolecule (e.g., a cationic polymer) form acomplex through ionic interactions. A therapeutic molecule (e.g., atherapeutic nucleic acid) and a cationic macromolecule (e.g., a cationicpolymer) may also be formed through covalent interactions.

In some embodiments, a complex comprising a therapeutic molecule (e.g.,a therapeutic nucleic acid) and a cationic macromolecule (e.g., acationic polymer) also comprises a mucopenetrating substance asdescribed herein. The mucopenetrating substance may exhibit mucolyticactivity, for example. In some embodiments, a complex comprises atherapeutic molecule (e.g., a therapeutic nucleic acid), a cationicmacromolecule (e.g., a cationic polymer), a mucopenetrating substance,and a non-ionic emulsifier.

In some embodiments, a complex is produced by combining amucopenetrating substance with a therapeutic nucleic acid and a cationicpolymer in an amount sufficient to charge neutralize the therapeuticnucleic acid.

Compositions

It is to be understood that a composition as provided herein cancomprise any therapeutic molecule, any cationic macromolecule, and anymucopenetrating substance described herein. In some embodiments, acomposition comprises at least two mucopenetrating substances, forexample, a mucolytic substance and at least one other mucopenetratingsubstance. In some embodiments, a composition comprises a therapeuticmolecule, a cationic macromolecule, a mucopenetrating substance (e.g., amucolytic substance), and a non-ionic emulsifier. Non-limiting examplesof compositions contemplated herein are provided in Table 1.

TABLE 1 Non-limiting examples of therapeutic compositions for deliveryto the GI tract Mucopenetrating Substance Therapeutic Additional AgentNucleic Acid Cationic Polymer Mucolytic Non-ionic Emulsifier (optional)Antisense polyethylenimine bromalein oleoyl polyoxyl-6 oligonucleotide(PEI) glyceride/oleoyl (ASO) macrogol-6 glyceride (LABRAFIL ®) ASOMADQUAT bromalein oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6glyceride (LABRAFIL ®)

In some embodiments, a composition comprises an antisenseoligonucleotide (ASO), polyethylenimine (PEI), and a mucopenetratingsubstance, wherein the PEI is present in an amount sufficient to chargeneutralize the therapeutic nucleic acid.

In some embodiments, a composition comprises an ASO,poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT), and amucopenetrating substance, wherein the MADQUAT is present in an amountsufficient to charge neutralize the therapeutic nucleic acid.

The concentration of therapeutic molecule in a composition may vary,depending on the particular molecule and the intended therapeuticeffect. In some embodiments, the concentration of a therapeutic moleculeis 0.0001-1000 mg/ml.

The concentration of cationic macromolecule in a composition may vary,depending on the amount required to charge neutralize the therapeuticmolecule in the composition. In some embodiments, the concentration of acationic macromolecule is 0.0001-1000 mg/ml.

The concentration of a mucopenetrating substance in a composition mayvary. In some embodiments, the concentration of a mucopenetratingsubstance is 0.0001-1000 mg/ml.

The concentration of a non-ionic emulsifier in a composition may vary.In some embodiments, the concentration of a non-ionic emulsifier is0.0001-1000 mg/ml.

The ratio of any two substances in a composition may vary. For example,the ratio of any two substances (e.g., cationic macromolecule andtherapeutic molecule, cationic molecule and mucopenetrating substance,cationic macromolecule and non-ionic emulsifier, therapeutic moleculeand mucopenetrating substance, therapeutic molecule and non-ionicemulsifier, or mucopenetrating substance and non-ionic emulsifier) maybe at least 1:1 to at least 10:1 (e.g., at least 1:1, at least 2:1, atleast 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, atleast 8:1, at least 9:1, or at least 10:1).

In some embodiments, a composition comprises a cationic macromolecule(e.g., polymer) and a therapeutic molecule (e.g., nucleic acid) at aratio sufficient to charge neutralize the therapeutic molecule. In someembodiments, a cationic macromolecule and the therapeutic molecule arepresent at a ratio of at least 1:1 (e.g., at least 1:1, at least 2:1, atleast 3:1, at least 4:1, at least 5:1, or at least 10:1) cationicmacromolecule:therapeutic molecule. In some embodiments, a cationicmacromolecule and the therapeutic molecule are present at a ratio ofmore than 10:1 (e.g., at least 12:1, at least 15:1, at least 20:1, atleast 50:1, or at least 100:1) cationic macromolecule:therapeuticmolecule.

In some embodiments, a composition comprises a cationic macromolecule(e.g., polymer) a therapeutic molecule (e.g., nucleic acid), and amucopenetrating substance. In some embodiments, the mucopenetratingsubstance and the therapeutic molecule and are present at a ratio of atleast 1:1 (e.g., at least 1:1, at least 2:1, at least 3:1, at least 4:1,at least 5:1, or at least 10:1) mucopenetrating substance:therapeuticmolecule. In some embodiments, a cationic macromolecule and thetherapeutic molecule are present at a ratio of more than 10:1 (e.g., atleast 12:1, at least 15:1, at least 20:1, at least 50:1, or at least100:1 mucopenetrating substance:therapeutic molecule.

Compositions comprising therapeutic nucleic acids may be formulated, forexample, as a solid or a liquid for oral, rectal, gastric, or vaginaldelivery. In some embodiments, a composition is formulated as a solidtablet or a lyophilized powder. In some embodiments, a solid dosage formhas a protective coating (e.g., an enteric coating). Non-limitingexamples of protective coatings include methyl acrylate-methacrylic acidcopolymers, cellulose acetate phthalate (CAP), cellulose acetatesuccinate, hydroxypropyl methyl cellulose phthalate, hydroxypropylmethyl cellulose acetate succinate (hypromellose acetate succinate),polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acidcopolymers, shellac, cellulose acetate trimellitate, sodium alginate,and zein. In some embodiments, a composition is formulated as aslow-release composition.

Composition herein may further comprise a pharmaceutically acceptableexcipient (e.g., carrier, buffer, and/or salt, etc.). A molecule orother substance/agent is considered “pharmaceutically acceptable” if itis approved or approvable by a regulatory agency of the Federalgovernment or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, includinghumans. An excipient may be any inert (inactive), non-toxic agent,administered in combination with a therapeutic molecule. Non-limitingexamples of excipients include buffers (e.g., sterile saline), salts,carriers, preservatives, fillers, coloring agents.

Cells and Tissues

Provided here are cells comprising any one of the compositionscomprising therapeutic nucleic acids described herein. In someembodiments, the cell is a gastrointestinal tract cell. For example, acell may be an intestinal epithelial cell that lines the surface ofintestinal epithelium. A cell as provided herein may be an isolatedcell, part of a tissue, or present in a subject (e.g., in the GI tractof a subject or model organism). In some embodiments, a cell is a humancell, a pig cell, or a rodent cell.

Methods

Any of the compositions described herein may be delivered to a subject,for example, to treat a gastrointestinal disorder (e.g., disease). Insome embodiments, the gastrointestinal disorder is an inflammatory boweldisorder. Non-limiting examples of inflammatory bowel disorders includeirritable bowel syndrome (IBS), ulcerative colitis, and Crohn's disease.

A subject, in some embodiments, is a human subject. Other mammaliansubjects are contemplated herein. For example, a subject may be aveterinary subject (e.g., cat, dog, horse, cow, sheep, pig, etc.).

The route of delivery may be oral, nasal, intravenous, subcutaneous,intramuscular, or intraperitoneal. Other routes of delivery arecontemplated herein. In some embodiments, the route of delivery is oral,for example, a composition is formulated as a enteric-coated table. Insome embodiments, a composition is delivered, directly or indirectly, toa mucosal surface of a subject (e.g., mucosal layer lining the GItract).

The methods herein encompass delivery of a single composition ordelivery of multiple composition, simultaneously or successively. Forexample, a method herein may include delivering a subject a compositioncomprising a therapeutic molecule and cationic polymer and alsodelivering to the subject a composition comprising a mucopenetratingsubstance and/or a non-ionic emulsifier.

In some embodiments, delivery of a composition comprising a therapeuticmolecule, such as a therapeutic nucleic acid, results in a decrease ingene expression in a cell of the subject. Thus, a therapeutic nucleicacid may target a gene of interest and inhibit expression of that gene,for example, by binding to the gene or the mRNA encoded by the gene.Thus, in some embodiments, a method comprises delivering to a subject acomposition comprising a mucopenetrating substance, a therapeuticnucleic acid, and a cationic polymer in an amount sufficient to chargeneutralize the therapeutic nucleic acid, wherein delivery of thecomposition decreases gene expression in a cell of the subject, relativeto baseline expression of the gene (not exposed to the therapeuticnucleic acid) or relative to expression of the gene following deliveryof a control composition with the therapeutic nucleic acid but withoutthe cationic macromolecule and/or mucopenetrating substance. In someembodiments, gene expression is decreased by at least 10%, at least 20%,at least 30%, at least 40%, or at least 50%, relative to a control.

Also provided herein are methods for synergistically decreasing geneexpression in a subject, comprising delivering to a mucosal surface of asubject a C10 fatty acid and a composition comprising a mucopenetratingsubstance, a therapeutic nucleic acid, and a cationic polymer in anamount sufficient to charge neutralize the therapeutic nucleic acid,wherein delivery of the composition synergistically decreases geneexpression in a cell of the subject. In some embodiments, thecomposition further comprises the C10 fatty acid. Synergy refers to theinteraction or cooperation of two or more substances to produce acombined effect greater than the sum of their separate effects. In someembodiments, gene expression is decreased by at least 10%, at least 20%,at least 30%, at least 40%, or at least 50%, relative to a control.

Devices

Computational oral physiologically-based pharmacokinetic (PBPK) modelsare used to predict oral bioavailability of therapeutic nucleic acidsand formulations thereof. See Lin and Harve, Pharmaceutics. 2017 Sep.26; 9(4). Pre-clinical methods to confirm or refute predictions based onPBPK models are of great significance as results obtained at thepre-clinical stage can help save resources at the clinical stage.Provided herein are preclinical assays and assay systems to assess themucopenetrability and/or enteric absorption of therapeutic nucleicacids.

Some aspects of the present disclosure provide a multiple well plate,wherein each well of the plate comprises a receiver chamber underlying apermeable membrane onto which a mucus layer has been deposited.

Some aspects of the present disclosure provide methods for assessingmucotransport of a substance, comprising applying the substance to awell described herein, and assessing transport of the substance throughthe mucus layer.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever.

Additional Embodiments

Additional embodiments of the present disclosure are encompassed by thefollowing numbered paragraphs:

1. A composition comprising a mucopenetrating substance, a therapeuticnucleic acid, and a cationic polymer in an amount sufficient to chargeneutralize the therapeutic nucleic acid.

2. A composition comprising an antisense oligonucleotide (ASO),polyethylenimine (PEI), and optionally a mucopenetrating substance,wherein the PEI is present in an amount sufficient to charge neutralizethe therapeutic nucleic acid.

3. A composition comprising an antisense oligonucleotide (ASO),poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT), and optionally amucopenetrating substance, wherein the MADQUAT is present in an amountsufficient to charge neutralize the therapeutic nucleic acid.

4. A composition comprising an antisense oligonucleotide (ASO),non-ionic emulsifier, and a cationic polymer, wherein the compositioncomprises the cationic polymer in an amount sufficient to chargeneutralize the ASO.

5. A composition comprising a therapeutic nucleic acid, a non-ionicemulsifier, and a cationic polymer having a molecular weight of 50 kDaor lower, wherein the composition comprises the cationic polymer in anamount sufficient to charge neutralize the ASO.

6. A composition comprising an antisense oligonucleotide (ASO),non-ionic emulsifier, and a zwitterionic polymer.

7. The composition of any one of the preceding numbered paragraphs,wherein the mucopenetrating substance is a non-ionic emulsifier and/orhas mucolytic activity.

8. The composition of any one of the preceding numbered paragraphs,wherein the therapeutic nucleic acid and the cationic polymer form acomplex through ionic interactions.

9. The composition of any one of the preceding numbered paragraphs,wherein the complex further comprises the mucopenetrating substance.

10. The composition of any one of the preceding numbered paragraphs,wherein the composition comprises at least two or at least threemucopenetrating substances.

11. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is a linear polymer.

12. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is a branched polymer.

13. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer comprises a cationic lipid.

14. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is selected from the group consisting of:polyquaternium, PDMAEMA (poly(2-dimethylaminoethyl methacrylate),MADQUAT (poly(2-(trimethylamino)ethyl methacrylate)), polyallylamines,polyvinylamines, polyethylenimine, polylysines, cationic polyaminoacids,and cationic polysaccharides.

15. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is selected from the group consisting of:polyallylamines, polyethyleneimines, and polylysines.

16. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is a polyethyleneimine.

17. The composition of any one of the preceding numbered paragraphs,wherein the polyethyleneimine is a branched polyethyleneimine.

18. The composition of any one of the preceding numbered paragraphs,wherein the polyethyleneimine has a molecular weight of about 10-25kilodaltons (kDa).

19. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is a polyallylamine.

20. The composition of any one of the preceding numbered paragraphs,wherein the polyallylamine has a molecular weight of lower than 50 kDa.

21. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer is a polylysine.

22. The composition of any one of the preceding numbered paragraphs,wherein the polylysine has a molecular weight of about 15-50 kDa.

23. The composition of any one of the preceding numbered paragraphs,wherein the concentration of cationic polymer in the composition isabout 10-30 mg/ml.

24. The composition of any one of the preceding numbered paragraphs,wherein the non-ionic emulsifier is selected from the group consistingof: polysorbates, poloxamers, polyoxylglycerides, macrogolglycerolricinoleate, polyethylene monostearate, sorbitan monoesters andtriesters, substituted polyethylene glycols, and derivative thereof.

25. The composition of any one of the preceding numbered paragraphs,wherein the non-ionic emulsifier is caprylocaproyl polyoxyl-8 glyceride(LABRASOL®), polysorbate 40 (TWEEN® 40), polysorbate 80 (TWEEN® 80),macrogolglycerol ricinoleate (KOLLIPHOR® P188), or oleoyl polyoxyl-6glyceride/oleoyl macrogol-6 glyceride) (LABRAFIL®).

26. The composition of any one of the preceding numbered paragraphs,wherein the concentration of non-ionic emulsifier in the composition isabout 10-40 mg/ml.

27. The composition of any one of the preceding claims, wherein themucopenetrating substance is selected from the group consisting of:bromohexine, L-cysteine methylester, bromalein, ambroxol, guaifenesin,and N-acetyl L-cysteine and dornase alfa, optionally wherein themucopenetrating substance is bromalein or decanoic acid.

28. The composition of any one of the preceding numbered paragraphs,wherein the mucopenetrating substance is bromalein and/or the non-ionicemulsifier is oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6glyceride)(LABRAFIL®).

29. The composition of any one of the preceding numbered paragraphs,wherein the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), optionally wherein the nucleic acid has a phosphorothioatebackbone.

30. The composition of any one of the preceding numbered paragraphs,wherein the nucleic acid is double-stranded or single-stranded.

31. The composition of any one of the preceding numbered paragraphs,wherein the nucleic acid comprises a modification, optionally a chemicalmodification and/or a genetic modification.

32. The composition of any one of the preceding numbered paragraphs,wherein the nucleic acid has a length of about 10 to 50 nucleotides.

33. The composition of any one of the preceding numbered paragraphs,wherein the nucleic acid is not a locked nucleic acid (LNA) or a peptidenucleic acid (PNA).

34. The composition of any one of the preceding numbered paragraphs,wherein the therapeutic nucleic acid is selected from the groupconsisting of antisense oligonucleotides and RNA interference molecules.

35. The composition of any one of the preceding numbered paragraphs,wherein the RNA interference molecules are selected from the groupconsisting of short-hairpin RNAs (shRNAs), small-interfering RNAs(siRNAs), and micro RNAs (mRNAs).

36. The composition of any one of the preceding numbered paragraphs,wherein the therapeutic nucleic acid is an antisense oligonucleotide(ASO).

37. The composition of any one of the preceding numbered paragraphs,wherein the therapeutic nucleic acid targets SMAD7 mRNA.

38. The composition of any one of the preceding numbered paragraphs,wherein the therapeutic nucleic acid is mongersen (GED-0301).

39. The composition of any one of the preceding numbered paragraphs,wherein the cationic polymer and the therapeutic nucleic acid arepresent at a ratio of at least 1:1, at least 5:1, or at least 10:1cationic polymer:therapeutic nucleic acid.

40. The composition of any one of the preceding numbered paragraphs,wherein the ASO is mongersen (GED-0301).

41. The composition of any one of the preceding numbered paragraphs,wherein the composition is a pharmaceutical composition furthercomprising a pharmaceutically-acceptable excipient.

42. The composition of any one of the preceding numbered paragraphs,wherein the therapeutic nucleic acid is an engineered nucleic acid,optionally a recombinant nucleic acid or a synthetic nucleic acid.

43. A cell comprising the composition of any one of the precedingnumbered paragraphs.

44. A complex produced by combining a mucopenetrating substance with atherapeutic nucleic acid, and a cationic polymer in an amount sufficientto charge neutralize the therapeutic nucleic acid.

45. A method comprising delivering to a subject the composition of anyone of the preceding numbered paragraphs.

46. A method comprising delivering to a subject a mucopenetratingsubstance, a therapeutic nucleic acid, and a cationic polymer in anamount sufficient to charge neutralize the therapeutic nucleic acid.

47. The method of paragraph 45 or 46, wherein the delivering is to amucosal surface of the subject.

48. A method for decreasing gene expression in a subject, comprisingdelivering to a mucosal surface of a subject the composition of any oneof the preceding numbered paragraphs, in an effective amount to decreasegene expression in a cell in a local region of the mucosal surface.

49. A method for synergistically decreasing gene expression in asubject, comprising delivering to a mucosal surface of a subject a C10fatty acid and the composition of any one of the preceding numberedparagraphs, in an effective amount to synergistically decrease geneexpression in a cell in a local region of the mucosal surface,optionally wherein the composition further comprises the C10 fatty acid.

50. The method of any one of the preceding numbered paragraphs, whereingene expression in the subject is reduced by at least 20% relative togene expression in a subject relative to gene expression in a subjectwho has not received the composition or has received a compositioncomprising the therapeutic nucleic acid without the cationic polymerand/or the mucopenetrating substance.

51. The method of any one of the numbered paragraphs, wherein thedelivering comprises orally, rectally, or vaginally delivering.

52. The method of any one of the numbered paragraphs, wherein thecomposition is in a solution, is lyophilized, or is in the form of atablet, optionally with an enteric coating.

53. The method of any one of the numbered paragraphs, wherein themucosal surface is the gastrointestinal tract, rectal tissue, or vaginaltissue.

54. The method of any one of the numbered paragraphs, wherein thesubject has a gastrointestinal disorder and/or has a compromisedgastrointestinal barrier.

55. The method of any one of the numbered paragraphs, wherein thegastrointestinal disorder is an inflammatory bowel disorder, optionallyirritable bowel syndrome (IBS), ulcerative colitis, or Crohn's disease.

56. The method of any one of the numbered paragraphs, wherein transportof the therapeutic nucleic acid through the mucosal surface is at least5-fold, at least 10-fold, at least 15-fold, or at least 20-fold higherthan uptake of a therapeutic nucleic acid without the cationic polymerand/or the mucopenetrating substance.

57. A multiple well plate, wherein each well of the plate comprises areceiver chamber underlying a permeable membrane onto which a mucuslayer has been deposited.

58. A method for assessing mucotransport of a substance, comprisingapplying the substance to a well of paragraph 57, and assessingtransport of the substance through the mucus layer.

59. The method of any one of the numbered paragraphs, wherein thesubstance comprises a mucopenetrating substance.

60. The method of any one of the numbered paragraphs, wherein thesubstance is a composition of any one of the preceding numberedparagraphs.

61. The composition of any one of the numbered paragraphs, wherein thecationic polymer is selected from polyallylamine (PALL), polylysine(PLL), and polyethyleneimine (PEI).

62. The composition of any one of the numbered paragraphs, wherein thecationic polymer is PALL.

63. The composition of any one of the numbered paragraphs, wherein thePALL has a molecule weight of lower than 50 kilodaltons (kDa).

64. The composition of any one of the numbered paragraphs 3, wherein thePALL has a molecular weight of about 10-20 kDa, optionally about 15 kDa.

65. The composition of any one of the numbered paragraphs, wherein thecationic polymer is PLL.

66. The composition of any one of the numbered paragraphs, wherein thePLL has a molecule weight of about 15-50 kDa.

67. The composition of any one of the numbered paragraphs, wherein thecationic polymer is PEI.

68. The composition of any one of the numbered paragraphs, wherein thePEI has a molecule weight of about 10-25 kDa.

69. The composition of any one of the numbered paragraphs, wherein thecationic polymer is branched.

70. The composition of any one of the numbered paragraphs, wherein theconcentration of the cationic polymer in the composition is about 10-30mg/ml.

71. The composition of any one of the numbered paragraphs, wherein thenon-ionic emulsifier is selected from oleoyl polyoxyl-6 glyceride/oleoylmacrogol-6 glyceride (LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN®40), polysorbate 80 (TWEEN® 80), and Kolliphor P188.

72. The composition of any one of the numbered paragraphs, wherein theconcentration of the non-ionic emulsifier is about 10-40 mg/ml.

73. The composition of any one of the numbered paragraphs, wherein thecationic polymer is PALL, optionally having a molecule weight of below50 kDa, and the non-ionic emulsifier is selected from oleoyl polyoxyl-6glyceride/oleoyl macrogol-6 glyceride (LABRAFIL®), Pluronic F127,polysorbate 40 (TWEEN® 40), polysorbate 80 (TWEEN® 80), and KolliphorP188.

74. The composition of any one of the numbered paragraphs, wherein thecationic polymer is PLL, optionally having a molecule weight of about15-50 kDa, and the non-ionic emulsifier is selected from oleoylpolyoxyl-6 glyceride/oleoyl macrogol-6 glyceride (LABRAFIL®), PluronicF127, polysorbate 40 (TWEEN® 40), polysorbate 80 (TWEEN® 80), andKolliphor P188.

75. The composition of any one of the numbered paragraphs, wherein thecationic polymer is PEI, optionally branched PEI, optionally having amolecule weight of 10-25 kDa, and the non-ionic emulsifier is selectedfrom oleoyl polyoxyl-6 glyceride/oleoyl macrogol-6 glyceride(LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN® 40), polysorbate 80(TWEEN® 80), and Kolliphor P188.

76. The composition of any one of the numbered paragraphs, wherein thecationic polymer has a molecular weight of about 10-50 kDa.

77. The composition of any one of the numbered paragraphs, wherein thecationic polymer has a molecular weight of about 15-50 kDa.

78. The composition of any one of the numbered paragraphs, wherein thecationic polymer has a molecular weight of about 10-25 kDa.

79. The composition of any one of the numbered paragraphs, wherein thetherapeutic nucleic acid is an antisense oligonucleotide (ASO).

81. The composition of any one of the numbered paragraphs, wherein thezwitterionic polymer is polyvinylpyrrolidine.

82. The composition of any one of the numbered paragraphs, wherein thepolyvinylpyrrolidine has a molecular weight of about 50-100 kDa.

EXAMPLES Example 1: Testing in Ex Vivo Pig Models Using Fluorescence InSitu Quantification

FIG. 1 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers at different concentrationscomplexed to FAM-Mongersen. Results are summarized as a heatmaps thatshows fold change relative to non-formulated Mongersen. White colorindicates fold changes higher than 4-fold.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. 27 uL of cationic polymers wasadded at concentration of 100 mg/ml (1:1), 30 mg/ml (1:3), and 10 mg/ml(1:10) in ddH20. For tissue transport experiments, formulations weredissolved in 100 uL PBS to mimic dissolution in jejunum, mixed bypipetting and then immediately used for porcine jejunum transportstudies. Transport studies were performed using a setup describedearlier (https://www.nature.com/articles/s41551-020-0545-6). The sampleswhere incubated for 1 hour, washed three times with PBS buffer followedby fluorescence intensity spectrophotometric analysis (M1000, Tecan) ofthe intact tissue. Experiments were performed with 4 replicates. Datawas analyzed by using Prism software (Graphpad, Version 8).

Testing various cationic polymers complexed with Mongersen showedincreased apical and basal tissue uptake compared to non-formulatedMongersen. The increase was both concentration dependent as well asdependent on the specific cationic polymer used.

FIG. 2 shows apical (left panel) and basal (right panel) jejunum tissueuptake of various formulations consisting of cationic polymers complexedto FAM-Mongersen combined with non-ionic emulsifiers at differentconcentrations. Results are summarized as a heatmaps that shows foldchange relative to non-formulated Mongersen. White color indicates foldchanges higher than 15-fold.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers wereadded at a ratio of 1:1 (10 uL of liquid emulsifier added to 10 uL 25microM FAM-labelled-mongersen), 3:1 (30 uL of liquid emulsifier added to10 uL 25 microM FAM-labelled-mongersen), and 5:1 (50 uL of liquidemulsifier added to 10 uL 25 microM FAM-labelled-mongersen). 27 uL ofcationic polymers was added at concentration of 100 mg/ml (1:1) inddH2O. For tissue transport experiments, formulations were dissolved in100 uL PBS to mimic dissolution in jejunum, mixed by pipetting and thenimmediately used for porcine jejunum transport studies. Transportstudies were performed using a setup described earlier(https://www.nature.com/articles/s41551-020-0545-6). The samples whereincubated for 1 hour, washed three times with PBS buffer followed byfluorescence intensity spectrophotometric analysis (M1000, Tecan) of theintact tissue. Experiments were performed with 4 replicates. Data wasanalyzed by using Prism software (Graphpad, Version 8).

The results show that apical and basal tissue uptake of cationicpolymer—Mongersen complexes can be further increased be the addition ofnon-ionic emulsifiers. The increase in fold changes is depended on bothconcentration and specific emulsifier and is specific to the cationicpolymer used.

FIG. 3 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with mucolytic agents at different concentrations. Results aresummarized as a heatmaps that shows fold change relative tonon-formulated Mongersen. White color indicates fold changes higher than20-fold.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Mucolytic agents were added ata ratio of 1:1 (10 uL of 100 mg/ml mucolytic agent in water added to 10uL 25 microM FAM-labelled-mongersen). 27 uL of cationic polymers wasadded at concentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine jejunum transport studies. Transport studies were performedusing a setup described earlier(https://www.nature.com/articles/s41551-020-0545-6). The samples whereincubated for 1 hour, washed three times with PBS buffer followed byfluorescence intensity spectrophotometric analysis (M1000, Tecan) of theintact tissue. Experiments were performed with 4 replicates. Data wasanalyzed by using Prism software (Graphpad, Version 8).

The results show that apical and basal tissue uptake of cationicpolymer—Mongersen complexes can be further increased be the additionmucolytic agents. The increase in fold changes is depended on bothconcentration and specific emulsifier and is specific to the cationicpolymer used. Absolute fold increases achieved are comparable withformulations containing cationic polymer-Mongersen complex withnon-ionic emulsifier.

FIGS. 4A-4B show Least Squares Means Plots, which show relative changein tissue permeability and apical tissue accumulation of FAM-Mongersenusing different molecular weight branched polyethyleneimine polymers.The results are based on a statistical regression analysis using 6different non-ionic emulsifiers combined withpolyethyleneimine-Mongersen polyplex.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Kolliphor P188, Labrafil, Tween 20, Tween 40, Tween 60, Tween 80) wereadded at a ratio of 1:1 (10 uL of liquid emulsifier added to 10 uL 25microM FAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine jejunum transport studies. Transport studies were performedusing a setup described earlier(https://www.nature.com/articles/s41551-020-0545-6). The samples whereincubated for 1 hour, washed three times with PBS buffer followed byfluorescence intensity spectrophotometric analysis (M1000, Tecan) of theintact tissue. Experiments were performed with 4 replicates. Data wasanalyzed by using JASP software.

The results show that apical tissue uptake and also transport across thetissue using PEI polymer—Mongersen complexes with non-ionic emulsifieris dependent on the molecular weight of PEI. The molecular weight thatis showing the highest increase in tissue uptake as well as permeationis PEI with an average molecular weight of 10-25 kDa. This trend is trueacross all different non-ionic emulsifier combinations tested.

Example 2: Testing in Ex Vivo Pig Models Using Immunohistochemistry(IHC) of Biopsy Samples

FIG. 5 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with additional excipients: Non-ionic emulsifiers (KolliphorP188, Labrafil, Tween 40, Tween 80), mycolytic (bromalein) orpermeability enhancer/mucodisruptor (decanoic acid). FAM fluorescenceintensity was quantified by fluorescence microscopy analysis of tissuecross-sections. The results show fold change relative to non-formulatedMongersen.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Kolliphor P188, Labrafil, Tween 40, Tween 80), mycolytic (bromalein) orpermeability enhancer/mucodisruptor (decanoic acid) were added at aratio of 1:1 (10 uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine jejunum transport studies. Transport studies were performedusing a setup described earlier(https://www.nature.com/articles/s41551-020-0545-6). Biopsy samples wereharvested after tissue was incubated for 1 hour and washed three timeswith PBS buffer followed by cyrosectioning and fluorescence microscopyanalysis of cryosections. The FAM fluorescence intensity of cyrosectionsat a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen. Quantification of fluorescence intensity based oncross-sections of jejunum tissue exposed to the formulations enables toselect for fluorescence signal within the GI tissue as opposed to signaloutside the tissue trapped in the mucus layer. Addition of additionalexcipients: Non-ionic emulsifier, mycolytic (bromalein) or permeabilityenhancer/mucodisruptor (decanoic acid) increased tissue uptake comparedto PEI polyplexes alone while the additional components were found toshow no improvement when formulated without the cationic agent PEI withFAM-Mongersen. This indicates a synergistic effect of the additionalexcipients in transport enhancement of FAM-Mongersen.

FIG. 6 shows apical and basal jejunum tissue uptake of variousformulations consisting of cationic polymers complexed to FAM-Mongersencombined with non-ionic emulsifiers including Kolliphor P188, Poloxamer407, Labrafil, Tween 20, Tween 40, Tween 60, Tween 80. FAM fluorescenceintensity was quantified by fluorescence microscopy analysis of tissuecross-sections. The results show fold change relative to non-formulatedMongersen.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Kolliphor P188, Labrafil, Tween 40, Tween 80), mycolytic (bromalein) orpermeability enhancer/mucodisruptor (decanoic acid) were added at aratio of 1:1 (10 uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS and then furtherdiluted by the following volume ratio in simulated intestinal fluid:1:2, 1:4, 1:20 to mimic dissolution and dilution occurring in jejunum.The formulations were mixed by pipetting and then immediately used forporcine jejunum transport studies. Transport studies were performedusing a setup described earlier(https://www.nature.com/articles/s41551-020-0545-6). Biopsy samples wereharvested after tissue was incubated for 1 hour and washed three timeswith PBS buffer followed by cyrosectioning and fluorescence microscopyanalysis of cryosections. The FAM fluorescence intensity of cyrosectionsat a magnification of 4×was quantified by ImageJ.

The results show transport enhancement of FAM-Mongersen PEI polyplexesis retained after SIF dilution up to a factor of 4 times dilutionsubstantially higher dilution of 20-fold results in no enhancement. Thisindicates that the formulation effect is dependent on the localconcentration of excipients exposed to the tissue shows a broadeffective concentration range.

FIG. 7 shows tissue uptake in esophagus, stomach, jejunum, colon andrectum of various formulations consisting of cationic polymers complexedto FAM-Mongersen combined with non-ionic emulsifiers (Labrasol and Tween40). FAM fluorescence intensity was quantified by fluorescencemicroscopy analysis of tissue cross-sections. The results show foldchange relative to non-formulated Mongersen in the corresponding tissuesegment.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Labrasol and Tween 40), were added at a ratio of 1:1 (10 uL of liquidemulsifier added to 10 uL 25 microM FAM-labelled-mongersen). 27 uL ofcationic polymers was added at concentration of 100 mg/ml (1:1) inddH20. For tissue transport experiments, formulations were dissolved in100 uL PBS to mimic dissolution in jejunum, mixed by pipetting and thenimmediately used for porcine GI tissue transport studies. Esophagus,stomach, jejunum, colon and rectum porcine tissue was used for thisstudy based on a setup described earlier(https://www.nature.com/articles/s41551-020-0545-6). Biopsy samples wereharvested after tissue was incubated for 1 hour and washed three timeswith PBS buffer followed by cyrosectioning and fluorescence microscopyanalysis of cryosections. The FAM fluorescence intensity of cyrosectionsat a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen across the different GI tissue segments suggesting that theseformulations could be effective to delivery oligonucleotides in variousGI segments other than jejunum. Interestingly, depending on theformulation and tissue segment the observed fold changes change bymultiple fold suggesting that each GI segment requires optimization offormulations to maximize transport.

Example 3: Testing in In Vivo Using Pig Models and Immunohistochemistry(IHC) of Biopsy Samples

FIG. 8 shows tissue uptake of various formulations consisting ofcationic polymers complexed to FAM-Mongersen combined with non-ionicemulsifiers (Labrasol and Tween 40) in jejunum. FAM fluorescenceintensity was quantified by fluorescence microscopy analysis of tissuecross-sections of biopsy samples harvested from jejunum segments exposedto formulations in pigs in vivo. The results show fold change relativeto non-formulated Mongersen in the corresponding tissue segment.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Labrasol and Tween 40 and Tween 80), were added at a ratio of 1:1 (10uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine GI tissue animal studies. For in vivo drug delivery studiesfemale Yorkshire pigs between 50 and 80 kg in weight were used. Beforeevery experiment, the animals were fasted overnight. On the day of theprocedure the morning feed was held. The animals were sedated with anintramuscular injection of telazol (tileramine/zolazepam) 5 mg/kg,xylazine 2 mg/kg and atropine 0.04 mg/kg. After complete sedation, thesmall intestine was accessed surgically. 1 cm jejunum segments werecreated by a custom device that compresses 0.5 cm of tissue on eitherside by tightly controlled magnetic force. 0.57 mL of formulation wasthen added in these segments via needle injection. After 1 hourincubation, the fluid was removed and the exposed tissue washed threetimes with PBS buffer followed by cyrosectioning and fluorescencemicroscopy analysis of cryosections. The FAM fluorescence intensity ofcyrosections at a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen in jejunum. This confirms the ex vivo results obtainedpreviously and validates the predictability of the system.

Example 4: Testing in Ex Vivo Pig Models Using Fluorescence In SituQuantification

FIGS. 9A-9B show effect of charged surfactants on tissue uptake offormulations. FIG. 9A shows tissue uptake of various formulationsconsisting of cationic polymers complexed to FAM-Mongersen combined withnon-ionic emulsifiers (Labrasol and Tween 40) in jejunum. FAMfluorescence intensity was quantified by fluorescence microscopyanalysis of tissue cross-sections of biopsy samples harvested fromjejunum segments exposed to formulations in pigs in vivo. FIG. 9B showsthe data in FIG. 9A in the form of a heatmap as show fold changerelative to non-formulated Mongersen in the corresponding tissuesegment.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Labrasol and Tween 40 and Tween 80), were added at a ratio of 1:1 (10uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine GI tissue animal studies. For in vivo drug delivery studiesfemale Yorkshire pigs between 50 and 80 kg in weight were used. Beforeevery experiment, the animals were fasted overnight. On the day of theprocedure the morning feed was held. The animals were sedated with anintramuscular injection of telazol (tileramine/zolazepam) 5 mg/kg,xylazine 2 mg/kg and atropine 0.04 mg/kg. After complete sedation, thesmall intestine was accessed surgically. 1 cm jejunum segments werecreated by a custom device that compresses 0.5 cm of tissue on eitherside by tightly controlled magnetic force. 0.57 mL of formulation wasthen added in these segments via needle injection. After 1 hourincubation, the fluid was removed and the exposed tissue washed threetimes with PBS buffer followed by cyrosectioning and fluorescencemicroscopy analysis of cryosections. The FAM fluorescence intensity ofcyrosections at a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen in jejunum. This confirms the ex vivo results obtainedpreviously and validates the predictability of the system.

FIGS. 10A-10B show IHC analysis of the fluorescence signal of Famlabeled Mongersen in vivo in pigs in the jejunum, where the Mongersen istreated with various Mongersen-polyplex formulations. effect of chargedsurfactants on tissue uptake of formulations.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Labrasol and Tween 40 and Tween 80), were added at a ratio of 1:1 (10uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine GI tissue animal studies. For in vivo drug delivery studiesfemale Yorkshire pigs between 50 and 80 kg in weight were used. Beforeevery experiment, the animals were fasted overnight. On the day of theprocedure the morning feed was held. The animals were sedated with anintramuscular injection of telazol (tileramine/zolazepam) 5 mg/kg,xylazine 2 mg/kg and atropine 0.04 mg/kg. After complete sedation, thesmall intestine was accessed surgically. 1 cm jejunum segments werecreated by a custom device that compresses 0.5 cm of tissue on eitherside by tightly controlled magnetic force. 0.57 mL of formulation wasthen added in these segments via needle injection. After 1 hourincubation, the fluid was removed and the exposed tissue washed threetimes with PBS buffer followed by cyrosectioning and fluorescencemicroscopy analysis of cryosections. The FAM fluorescence intensity ofcyrosections at a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen in jejunum. This confirms the ex vivo results obtainedpreviously and validates the predictability of the system.

FIGS. 11A-11B shows uptake of FAM-Mongersen into apical (FIG. 11A) andbasal (FIG. 11B) intestinal tissue for various formulations.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Labrasol and Tween 40 and Tween 80), were added at a ratio of 1:1 (10uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulation were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine GI tissue animal studies. For in vivo drug delivery studiesfemale Yorkshire pigs between 50 and 80 kg in weight were used. Beforeevery experiment, the animals were fasted overnight. On the day of theprocedure the morning feed was held. The animals were sedated with anintramuscular injection of telazol (tileramine/zolazepam) 5 mg/kg,xylazine 2 mg/kg and atropine 0.04 mg/kg. After complete sedation, thesmall intestine was accessed surgically. 1 cm jejunum segments werecreated by a custom device that compresses 0.5 cm of tissue on eitherside by tightly controlled magnetic force. 0.57 mL of formulation wasthen added in these segments via needle injection. After 1 hourincubation, the fluid was removed and the exposed tissue washed threetimes with PBS buffer followed by cyrosectioning and fluorescencemicroscopy analysis of cryosections. The FAM fluorescence intensity ofcyrosections at a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen in jejunum. This confirms the ex vivo results obtainedpreviously and validates the predictability of the system.

FIGS. 12A-12B shows uptake of FAM-Mongersen into apical (FIG. 11A) andbasal (FIG. 11B) intestinal tissue for various formulations with andwithout mucolytics.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(Labrasol and Tween 40 and Tween 80), were added at a ratio of 1:1 (10uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used forporcine GI tissue animal studies. For in vivo drug delivery studiesfemale Yorkshire pigs between 50 and 80 kg in weight were used. Beforeevery experiment, the animals were fasted overnight. On the day of theprocedure the morning feed was held. The animals were sedated with anintramuscular injection of telazol (tileramine/zolazepam) 5 mg/kg,xylazine 2 mg/kg and atropine 0.04 mg/kg. After complete sedation, thesmall intestine was accessed surgically. 1 cm jejunum segments werecreated by a custom device that compresses 0.5 cm of tissue on eitherside by tightly controlled magnetic force. 0.57 mL of formulation wasthen added in these segments via needle injection. After 1 hourincubation, the fluid was removed and the exposed tissue washed threetimes with PBS buffer followed by cyrosectioning and fluorescencemicroscopy analysis of cryosections. The FAM fluorescence intensity ofcyrosections at a magnification of 4×was quantified by ImageJ.

The results show increased transport of FAM-Mongersen into intestinaltissue of the PEI polyplexes tested compared to non-formulated FAMMongersen in jejunum. This confirms the ex vivo results obtainedpreviously and validates the predictability of the system.

Example 5: Analysis of Diffusion Through Native Porcine Mucus UsingTransWell System

FIG. 13 shows transport through native porcine mucus obtained from thejejunum of various FAM-Mongersen formulations. Microdiffusion iscalculated by measured FAM fluorescence intensity in receiver chambercompared to the initial donor fluorescence intensity after 1 hour ofincubation.

Samples were prepared as follows, FAM-labelled-mongersen was dissolvedin ddH2O at a concentration of 25 microM. Non-ionic emulsifiers(kolliphor P188, Tween 40 and Tween 80), were added at a ratio of 1:1(10 uL of liquid emulsifier added to 10 uL 25 microMFAM-labelled-mongersen). 27 uL of cationic polymers was added atconcentration of 100 mg/ml (1:1) in ddH20. For tissue transportexperiments, formulations were dissolved in 100 uL PBS to mimicdissolution in jejunum, mixed by pipetting and then immediately used formucus transport studies. For mucus transport studies, native mucus wascollected from freshly harvested jejunum segments from adult Yorkshirepigs, and 0.3 mL was added on 24 well plate polycarbonate Transwellinsert with 8 um pore size. The receiver chamber was filled with PBSbuffer until contact was reached with transwell filter. Then, 0.5 mL offormulation was carefully added on each transwell filter and incubatedfor 1 hour. Subsequently, 50 uL of the receiver fluid was removed andFAM fluorescence intensity measured via plate reader analysis.

The results reveal that transport across the mucus layer is increasedfor FAM-Mongersen-PEI polyplexes in presence of a non-ionic emulsifier.FAM-Mongersen-PEI polyplexes alone show poor mucus permeability. Thissuggests that the non-ionic emulsifier enhance overall jejunum transportby increasing mucus penetration.

Example 6: Gastrointestinal Tract Update with Other Oligonucleotides

FIG. 14 shows apical jejunum tissue uptake of various formulationsconsisting of cationic polymers complexed to Cy5-siRNA combined withnon-ionic emulsifiers. Tissue uptake was analyzed by measuring cy5fluorescent signal intensity of ex vivo jejunum tissue exposed withCy5-siRNA formulations for 1 hour. The results show fold change relativeto non-formulated Cy5-siRNA.

FIG. 15 shows apical jejunum tissue uptake of various formulationsconsisting of cationic polymers complexed to Cy3 conjugated plasmid DNAcombined with non-ionic emulsifiers. Plasmid DNA was complexed with ahigher and a lower concentration of cationic polymer (high, low). Tissueuptake was analyzed by measuring cy3 fluorescent signal intensity of exvivo jejunum tissue exposed with Cy3-Plasmid DNA formulations for 1hour. The results show fold change relative to non-formulatedCy3-Plasmid DNA.

Example 7: GI-ORIS Studies—Fluorescence-Based Experiments UsingFAM-Mongersen

The purpose of these studies was to evaluate of the effect of polymerarchitecture on antisense oligonucleotide (ASO) accumulation in jejunumtissue. Three (3) different cationic polymers (polyallylamine (PALL),polylysine (PLL), and polyethyleneimine (PEI)) as well as a zwitterionicpolymer (polyvinylpyrrolidine (PVP)) were tested in combination withdifferent non-ionic emulsifiers (Labrafil (LFCS), Pluronic F127,polysorbate 40 (TWEEN® 40) (T40) or polysorbate 80 (TWEEN® 80) (T80)).

The samples were prepared with the follow concentrations of reagents:fluorescein (FAM)-labeled-Mongersen ASO in phosphate buffered saline(PBS) (300 μg/mL); cationic polymer (34 or 68 mg/mL) in PBS; non-ionicemulsifier (2.8 mg/mL) in PBS. For each study, the sample formulationwas diluted by 25% to mimic dissolution in the jejunum. Transportstudies were performed as described elsewhere (von Erlach T et al.Nature Biomedical Engineering volume 4, pages 544-559 (2020)). Thesamples were incubated for 1 hour, washed multiple times with PBS, andfluorescence intensity spectrophotometric analysis (M1000, Tec an) ofthe intact tissue was performed. The experiments were performed with 8replicates. The data were analyzed using Prism software (Graphpad,Version 8) and Tableau software. Results are shown in FIGS. 16-19 .

The relative change in apical tissue accumulation of FAM-Mongersen usingthe different molecular weight branched polyethyleneimine polymers (1.2kilodaltons (kDa), 2 kDa, 10 kDa, 25 kDa, 70 kDa, or 750 kDa) wasanalyzed using a Least Squares Means Plot (FIG. 16 ). The results werebased on a statistical regression analysis using six (6) differentnon-ionic emulsifiers combined with a PEI-Mongersen polyplex.

The average apical tissue accumulation of FAM-Mongersen was determinedusing the different molecular weight polyallylamine polymers (FIG. 17 )or polylysine polymers (FIG. 18 ) combined with four (4) differentnon-ionic emulsifiers (Labrafil, Pluronic F127, polysorbate 40 (TWEEN®40) and polysorbate 80 (TWEEN® 80). Values are expressed as fold changecompared to the non-formulated FAM-labeled Mongersen control.

The most effective molecular weight ranges for each polymer andemulsifier tested are provided in Table 2.

TABLE 2 Most Effective Molecular Weights of Polymers Polymer EffectiveMolecular Weight Range Polyethyleneimine branched 10-25 kDaPolyallylamine Below 50 kDa Polylysine 16-50 kDa Polyvinylpyrrolidine50-100 kDa

With respect to polymer and emulsifier concentration, PALL was mosteffective at a concentration in the range of 11-28 mg/ml regardless ofemulsifier type/concentration. At that concentration, the molar ratiobetween PALL and FAM-Mongersen was 16 to 40. Likewise, both TWEEN® 80and Kolliphor P188 (K188) were effective at a concentration in the rangeof 14-36 mg/ml. Results are summarized as a bar graph in FIG. 19 , whichshows fold change relative to Mongersen in PBS buffer.

Example 8: LCMS Studies—Tissue Biopsies of the GI ORIS Tissue Plate

The purpose of these studies was to compare fluorescence signaldetection of FAM-labeled Mongersen accumulation in jejunum tissue usingmass spectrometry detection, relative to a non-labeled Mongersencontrol.

The samples were prepared with the follow concentrations of reagents:FAM-labeled Mongersen ASO in PBS (300 μg/mL); non-labeled Mongersen ASOin PBS (300 μg/mL). The Mongersen ASO was complexed to cationic polymersand emulsifiers and diluted with PBS to reach desired concentrations ofthe Mongersen ASO. Transport studies were performed as describedelsewhere (von Erlach T et al. Nature Biomedical Engineering volume 4,pages 544-559 (2020)). The samples were incubated for 1 hour, washedmultiple times with PBS, and fluorescence intensity spectrophotometricanalysis (M1000, Tec an) of the intact tissue was performed. Theexperiments were performed with 18 replicates for fluorescence and 3replicates for mass spectrometry. Tissue pieces were biopsied,homogenized and subsequently analyzed by mass spectrometry (Agilent110Agilent 1100 HPLC-UV/MS0).

Non-labeled Mongersen and FAM-labeled Mongersen showed similar resultsin relative changes of Mongersen jejunum tissue accumulation,demonstrating that fluorescence-based detection of FAM-labeled Mongersenis a suitable method to evaluate Mongersen jejunum tissue accumulation.

Table 3 provides a summary of the results, showing average Mongersenjejunum tissue accumulation fold change of PALL 15 kDa formulations withthe non-ionic emulsifiers Kolliphor P188 and TWEEN® 80, or TWEEN®80alone, compared to non-formulated control. Fold change of non-labeledMongersen tissue accumulation by mass spectrometry detection (LCMS) wascompared to fluorescence detection using FAM-labeled Mongersen (FL). Theratio between the fold changes of the two detection methods is shown forcomparison (Ratio).

TABLE 3 Summary of Results Fold Change Fold Change (relative to 160μg/mL (relative to 80 μg/mL Mongersen) Mongersen) Formulation LCMS FLRatio LCMS FL Ratio T80_28 1.4 0.9 1.5 0.7 0.8 0.9 PALL: 4.9 4.3 1.2 3.75.5 0.7 15-T80_8_28 PALL: 6.3 7.7 0.8 3.2 9.3 0.3 15-K188_17_28 PBS 1.01.0 1.0 1.0 1.0 1.0

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical valuemean±10% of the recited numerical value.

Where a range of values is provided, each value between the upper andlower ends of the range are specifically contemplated and describedherein.

What is claimed is:
 1. A composition comprising a mucopenetratingsubstance, a therapeutic nucleic acid, and a cationic polymer in anamount sufficient to charge neutralize the therapeutic nucleic acid. 2.The composition of claim 1, wherein the mucopenetrating substance is anon-ionic emulsifier.
 3. The composition of claim 1 or 2, wherein themucopenetrating substance has mucolytic activity.
 4. The composition ofany one of the preceding claims, wherein the therapeutic nucleic acidand the cationic polymer form a complex through ionic interactions. 5.The composition of claim 4, wherein the complex further comprises themucopenetrating substance.
 6. The composition of any one of thepreceding claims, wherein the composition comprises at least two or atleast three mucopenetrating substances.
 7. The composition of any one ofthe preceding claims, wherein the cationic polymer is a linear polymeror a branched polymer.
 8. The composition of any one of the precedingclaims, wherein the cationic polymer comprises a cationic lipid.
 9. Thecomposition of any one of the preceding claims, wherein the therapeuticnucleic acid is an antisense oligonucleotide (ASO), optionally mongersen(GED-0301).
 10. A composition comprising an antisense oligonucleotide(ASO), non-ionic emulsifier, and a cationic polymer, wherein thecomposition comprises the cationic polymer in an amount sufficient tocharge neutralize the ASO.
 11. The composition of any one of thepreceding claims, wherein the cationic polymer is selected frompolyallylamine (PALL), polylysine (PLL), and polyethyleneimine (PEI).12. The composition of claim 11, wherein the cationic polymer is PALL.13. The composition of claim 12, wherein the PALL has a molecule weightof lower than 50 kilodaltons (kDa).
 14. The composition of claim 13,wherein the PALL has a molecular weight of about 10-20 kDa, optionallyabout 15 kDa.
 15. The composition of claim 11, wherein the cationicpolymer is PLL.
 16. The composition of claim 15, wherein the PLL has amolecule weight of about 15-50 kDa.
 17. The composition of claim 11,wherein the cationic polymer is PEI.
 18. The composition of claim 17,wherein the PEI has a molecule weight of about 10-25 kDa.
 19. Thecomposition of claim 18, wherein the cationic polymer is branched. 20.The composition of any one of claims 10-19, wherein the concentration ofthe cationic polymer in the composition is about 10-30 mg/ml.
 21. Thecomposition of any one of the preceding claims, wherein the non-ionicemulsifier is selected from oleoyl polyoxyl-6 glyceride/oleoylmacrogol-6 glyceride (LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN®40), polysorbate 80 (TWEEN® 80), and Kolliphor P188.
 22. The compositionof any one of any one of the preceding claims, wherein the concentrationof the non-ionic emulsifier is about 10-40 mg/ml.
 23. The composition ofany one of any one of claim 1-10, wherein the cationic polymer is PALL,optionally having a molecule weight of below 50 kDa, and the non-ionicemulsifier is selected from oleoyl polyoxyl-6 glyceride/oleoylmacrogol-6 glyceride (LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN®40), polysorbate 80 (TWEEN® 80), and Kolliphor P188.
 24. The compositionof any one of claims 1-10, wherein the cationic polymer is PLL,optionally having a molecule weight of about 15-50 kDa, and thenon-ionic emulsifier is selected from oleoyl polyoxyl-6 glyceride/oleoylmacrogol-6 glyceride (LABRAFIL®), Pluronic F127, polysorbate 40 (TWEEN®40), polysorbate 80 (TWEEN® 80), and Kolliphor P188.
 25. The compositionof any one of claims 1-10, wherein the cationic polymer is PEI,optionally branched PEI, optionally having a molecule weight of 10-25kDa, and the non-ionic emulsifier is selected from oleoyl polyoxyl-6glyceride/oleoyl macrogol-6 glyceride (LABRAFIL®), Pluronic F127,polysorbate 40 (TWEEN® 40), polysorbate 80 (TWEEN® 80), and KolliphorP188.
 26. A composition comprising a therapeutic nucleic acid, anon-ionic emulsifier, and a cationic polymer having a molecular weightof 50 kDa or lower, wherein the composition comprises the cationicpolymer in an amount sufficient to charge neutralize the ASO.
 27. Thecomposition of claim 26, wherein the cationic polymer has a molecularweight of about 10-50 kDa.
 28. The composition of claim 27, wherein thecationic polymer has a molecular weight of about 15-50 kDa.
 29. Thecomposition of claim 28, wherein the cationic polymer has a molecularweight of about 10-25 kDa.
 30. The composition of any one of thepreceding claims, wherein the cationic polymer and the therapeuticnucleic acid are present at a ratio of at least 1:1, at least 5:1, or atleast 10:1 cationic polymer:therapeutic nucleic acid.
 31. Thecomposition of any one of the foregoing claims, wherein the compositionis a pharmaceutical composition further comprising apharmaceutically-acceptable excipient.
 32. The composition of any one ofthe foregoing claims, wherein the therapeutic nucleic acid is anengineered nucleic acid, optionally a recombinant nucleic acid or asynthetic nucleic acid.
 33. A cell comprising the composition of any oneof the preceding claims.
 34. A complex produced by combining amucopenetrating substance, a therapeutic nucleic acid, and a cationicpolymer in an amount sufficient to charge neutralize the therapeuticnucleic acid.
 35. A method comprising delivering to a subject thecomposition of any one of the preceding claims.
 36. A method comprisingdelivering to a subject a mucopenetrating substance, a therapeuticnucleic acid, and a cationic polymer in an amount sufficient to chargeneutralize the therapeutic nucleic acid.
 37. The method of claim 35 or36, wherein the delivering is to a mucosal surface of the subject.
 38. Amethod for decreasing gene expression in a subject, comprisingdelivering to a mucosal surface of a subject the composition of any oneof the preceding claims, in an effective amount to decrease geneexpression in a cell in a local region of the mucosal surface.
 39. Amethod for synergistically decreasing gene expression in a subject,comprising delivering to a mucosal surface of a subject a C10 fatty acidand the composition of any one of the preceding claims, in an effectiveamount to synergistically decrease gene expression in a cell in a localregion of the mucosal surface, optionally wherein the compositionfurther comprises the C10 fatty acid.
 40. The method of any one of thepreceding claims, wherein gene expression in the subject is reduced byat least 20% relative to gene expression in a subject relative to geneexpression in a subject who has not received the composition or hasreceived a composition comprising the therapeutic nucleic acid withoutthe cationic polymer and/or the mucopenetrating substance.
 41. Themethod of any one of the preceding claims, wherein the mucosal surfaceis the gastrointestinal tract, rectal tissue, or vaginal tissue.
 42. Themethod of any one of the preceding claims, wherein the subject has agastrointestinal disorder and/or has a compromised gastrointestinalbarrier.
 43. The method of any one of the preceding claims, wherein thegastrointestinal disorder is an inflammatory bowel disorder, optionallyirritable bowel syndrome (IBS), ulcerative colitis, or Crohn's disease.44. A multiple well plate, wherein each well of the plate comprises areceiver chamber underlying a permeable membrane onto which a mucuslayer has been deposited.
 45. A method for assessing mucotransport of asubstance, optionally a mucopenetrating substance, comprising applyingthe substance to a well of claim 44, and assessing transport of thesubstance through the mucus layer.
 46. A composition comprising anantisense oligonucleotide (ASO), non-ionic emulsifier, and azwitterionic polymer, optionally a polyvinylpyrrolidine optionallyhaving a molecular weight of about 50-100 kDa.